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Aim: To validate a method for determining equivalent drug cutoff concentrations for tetrahydrocannabinol and amphetamine in blood and oral fluid, which ensures that the drug prevalence in samples of blood and oral fluid taken simultaneously is equal., Methods: A method using regression analysis of drug concentrations for defined percentiles in blood and oral fluid was developed. The accuracy and precision of this technique was investigated. As study populations, 311 cannabis users and 197 amphetamine users from the Rosita-2 Project were used., Results: A total of 80 paired oral fluid and blood concentrations were needed to determine accurate regression formulae. When using the formulae to calculate drug cutoff concentrations in oral fluid corresponding to 2.0, 4.0, 6.0, 8.0 and 10.0 ng/ml tetrahydrocannabinol in blood and 200, 400, 600, 800 and 1000 ng/ml amphetamine in blood, the accuracy was better than 100 ± 20% compared to actual prevalence in blood with precision better than ± 20%., Conclusion: Prevalence regression may be a useful tool in estimating equivalent cutoff concentrations in blood and oral fluid., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A fast and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of amphetamines (amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, ephedrine, and p-methoxyamphetamine) in plasma has been developed and validated. Sample preparation was performed by liquid-liquid extraction using ethyl acetate. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing all analytes at the column inlet, a gradient start, with acid mobile phase consisting of 0.1% formic acid and methanol was chosen. Positive electrospray ionization MS-MS detection was performed with two multiple reaction monitoring transitions for each analyte. Deuteriumlabeled internal standards were used for five of the analytes. The limit of detection was in the range 0.25-1.25 ng/mL, and the limit of quantification was fixed at the lowest calibrator of 2.5 ng/mL for all of the compounds. The RSD values of the intra- and interassay precision and accuracy were lower than 11% at four concentration levels, including two external quality controls. No or only minor matrix effects were observed, and the extraction method presented recoveries higher than 93% for all the compounds. Total run time, including equilibration, was 12 min. The method is routinely used at the National Institute of Criminalistics and Criminology for quantitative determination of the main amphetamines in plasma from forensic and driving under the influence cases.

Amphetamines and methadone are synthetic chiral drugs with a high potential for abuse. As such, several analytical methods have been developed for their enantioseparation and analysis in biological tissues, and some of these are based on electrodriven techniques. In this review, the most important and recent of these latter methods are reviewed and their main advantages and disadvantages are discussed. Particular attention is paid to the suitability of each method for the application to the biological matrix of interest: while all methods have been successfully applied for one or more biological tissues, to reach this goal they must overcome the sensitivity problem that is common to almost all capillary electrophoretic techniques. Most methods use one or more cyclodextrin derivatives as the chiral selector, thus the separation mechanism is not particularly complicated or unusual., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: To examine the associations between socio-economic characteristics and driving under the influence of drugs (DUID), and to elaborate determinants of drugged driving., Design: A register-based case-control study., Setting: Finland., Participants: Cases (n = 5859) apprehended by the police and suspected of DUID during 1993-2006 and controls (n = 74 809) drawn from the general Finnish population., Measurements: The effects of parents' and own education, urbanization of municipality, socio-economic position (SEP), main activity, income, marital status and living arrangements on DUID were estimated using logistic regression analysis. The analyses were conducted separately for men and women, age groups of under 45 years and aged 45 or over, and for substance groups of benzodiazepines only, benzodiazepines with alcohol, amphetamines and cannabinoids., Findings: Low education, unemployment, disability pension, being divorced and living alone were the strongest individual predictors of DUID in all substance groups. Illicit drug users were more disadvantaged compared to those in the benzodiazepines groups. Contrary to other substance and age groups, higher educational level and higher SEP were associated with DUID among benzodiazepine users aged 45 or over., Conclusions: A disadvantaged social background is a significant predictor of driving while under the influence of drugs for all substance use groups in Finland. The gradient is greater for amphetamines and cannabinoids than benzodiazepines., (© 2011 The Authors, Addiction © 2011 Society for the Study of Addiction.)

A rapid method has been developed to analyse morphine, codeine, 6-monoacetylmorphine, cocaine, benzoylecgonine, dihydrocodeine, cocaethylene, 3,4-methylenedioxyamphetamine, ketamine, 3,4-methylenedioxymethamphetamine, pseudoephedrine, lignocaine, benzylpiperazine, methamphetamine, amphetamine, methadone, phenethylamine and levamisole in human blood. Blood samples were cleaned up using mixed mode solid phase extraction using Evolute™ CX solid phase extraction cartridges and the sample aliquots were analysed by hybrid triple quadrupole linear ion trap (QTRAP) mass spectrometry with a runtime of 12.5 min. Multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification and confirmation was carried out by library search with a developed in-house MS/MS library based on EPI spectra at a collision energy spread of 35 ± 15 in positive mode and MRM ratios. The method was validated in blood, according to the criteria defined in Commission Decision 2002/657/EC. At least two MRM transitions for each substance were monitored in addition to EPI spectra. Deuterated analogues of analytes were used as internal standards for quantitation where possible. The method proved to be simple and time efficient and was implemented as an analytical strategy for the illicit drug monitoring of opioids, cocaines, amphetamines and adulterants in forensic cases of crime offenders, abusers or victims in the Republic of Ireland., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The authors examined benzodiazepine and sedative-hypnotic positive cases of drivers under the influence (DUI) in Finland from 1997 to 2008. Factors studied were the number of cases positive for the most commonly encountered of these pharmaceuticals, simultaneous use of amphetamine and/or cannabis, and the relationship between the number of DUI cases and overall sales in Finland for the individual pharmaceuticals. Data for 20037 cases positive for the relevant drugs were retrieved from the laboratory database of the Alcohol and Drug Analytics Unit, National Institute for Health and Welfare. Toxicological results were from blood analysis. Drug sales figures for each pharmaceutical were obtained from the Finnish Medicines Agency. An increase in DUI cases that were positive for the drugs studied was evident, which reflected the overall increase in positive DUI cases detected annually. The proportion of relevant cases was typically 75% or higher of all positive DUI cases up to 2003, the year that the Finnish zero tolerance law was introduced, and then decreased to 66.2% in 2008. Diazepam was consistently the most commonly detected nonmetabolite drug. The prevalence of clonazepam and alprazolam increased sharply from 2004 onward. Metabolites of diazepam, nordiazepam, temazepam, and oxazepam, were other common findings. Associated use of amphetamine and/or cannabis was also common in these DUI cases, typically between 56% and 66% of cases. An increase in the number of DUI cases showing combined use of benzodiazepines and sedative-hypnotics with amphetamines in particular was apparent after zero tolerance legislation and the introduction in 2005 of an effective on-site screening device for the stimulant. Ratios of DUI cases to sales figures showed an increase in detection of clonazepam-positive DUI cases from 2003. Diazepam, midazolam, and alprazolam also exhibited relatively high ratios, which increased from 1997 to 2008.

Two recent cases of death due to p-methoxyamphetamine (PAM) intake, a methoxylated phenethylamine derivative, were described and compared with previous PMA related deaths that occurred in many countries. Following a review of the available literature from 1974 to 2011 three periods of resurgence of an unsuspected increase of lethal PMA causation were considered. Signs of intoxications, concentrations of PMA found in biological materials were discussed. Based on the case reports can be concluded the great number of victims were unconscious of taking PMA as substitute of MDMA. Two new methods for screening, identification and quantification of amphetamine derivatives in biosamples (blood and urine) using LC-MS/MS techniques were developed. The methods have proven to be appropriate for clinical and forensic toxicology purposes.

We previously reported that treatment of prepubertal male rats with low, injected or oral, doses of methylphenidate stimulated cfos, fosB and arc expression in many areas of the developing brain. In the present study our objective was to determine whether the widely prescribed psychostimulant Adderall XR (ADD) exerted similar effects in infantile and prepubertal rat brain. We report here, for the first time, that low threshold doses of oral ADD, an extended-release mixture of amphetamine salts, now routinely used for the treatment of Attention Deficit Hyperactivity Disorder (ADHD), also increased cfos expression in infantile (postnatal day 10; PD10) and prepubertal (PD24) rat brain. These threshold doses were correlated with blood levels of amphetamine determined by liquid chromatography-mass spectrometry. Moreover, we observed that chronic treatment with oral ADD (1.6 mg/kg; x 14 days) not only significantly down-regulated cfos expression following a final challenge dose of ADD in prepubertal (PD24) rat striatum and cortex, quantified in terms of FOS immunoreactivity (FOS-ir), but did so at a daily dose that was without effect with methylphenidate (MPH); that is a much higher oral dose of MPH (7.5 mg/kg; x 14 days) failed to induce down-regulation of cfos expression. Similar experiments in infantile rats (PD10), but using a threshold injected dose of ADD (1.25 mg/kg sc) also significantly reduced striatal and cingulate cortical FOS-ir. An additional finding in the prepubertal rats was that oral ADD-induced FOS-ir was observed in the cerebral cortex following doses lower than the threshold dose necessary to increase FOS-ir in the striatum. This was not the case in the PD10 rats. In conclusion, our efforts to calibrate biological responses, such as immediate early gene expression, to clinically relevant blood levels of stimulants confirmed that expression of cfos is very sensitive to repeated low doses of Adderall XR. It is now feasible to examine whether other genes are also affected in these young rats and if the changes we report are reversible. The implications of such studies should be relevant to the putative effects of psychostimulant treatment of very young children., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

To investigate the changes of regional cerebral blood flow (rCBF) in early-onset anorexia nervosa (AN) before and after weight gain, we examined resting rCBF using single photon emission computed tomography (SPECT) with [(123)I]iodoamphetamine ((123)I-IMP). Ten female children with AN (mean age 13.2 years old) participated in this study. SPECT examinations were performed in all patients twice at the beginning of treatment and after weight gain. The mean body mass index (BMI) was changed from 13.1 to 16.6 during 4 months treatment period. Automatic voxel-based analysis of the images was carried out using statistical parametric mapping (SPM) software. Relatively increased rCBF in the bilateral parietal lobe and limbic lobe including the posterior cingulate cortex (PCC) were observed after weight gain in early-onset AN. There was no significant decrease in the rCBF after weight gain. A significant positive correlation was observed between BMI and rCBF in the right thalamus, right parietal lobe, and right cerebellum. These results suggested that weight gain during the process of recovery from early-onset AN might activate specific brain regions which are possibly relevant to the pathophysiological aspects of the disorder., (Copyright 2009 Elsevier B.V. All rights reserved.)

A retrospective study was carried out on 133 suicidal hanging cases autopsied at Forensic Medicine Center in Dammam, Kingdom of Saudi Arabia (from 2003 to 2007). Of these cases, 115 (86.46%) were males and 18 females (13.54%), with an average male:female ratio of 6.38. There was an increasing trend of hanging among ages between 21 and 50 years (88.7%), and the fourth decade had the highest number of victims (about 36%) between all age groups. Local Saudi nationals comprised a small proportion of cases (21 persons, 15.8%), while the others were foreigners working in the Kingdom with an Indian precedence (63 persons, 47.4%), followed by other 11 different nationalities representing 36.8% of the cases. The peak of these suicidal acts was in June and the least was in February and December. Postmortem blood alcohol was found in 6.76% of cases, amphetamine in 3% (4 cases), and cannabinoids in 2.25% (3 cases). About 75% of the deceased persons were male laborers (100 cases), 11.27% were female housemaids (15 cases), and only 13 persons were unemployed (9.77%). Previous history of psychological illnesses was documented in 19 cases (14.28%) and stressful family problems in 6% of cases. There were previous suicidal attempts in only one case. Suicidal note was found in only seven cases. In conclusion, there was a decreasing trend of suicide by hanging in Dammam from 39 cases in 2003 to 17 cases in 2007, with a rate of 0.85/100,000 population in the last year. This was inconsistent with the overall increase in suicide rate in Dammam when compared to the previous decade., (Copyright 2010 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

The Rapid Stat assay, a point-of-collection drug-testing device for detection of amphetamines, cannabinoids, cocaine, opiates, methadone, and benzodiazepines in oral fluid, was evaluated for cannabis and amphetamine-type stimulants. The Rapid Stat tests (n = 134) were applied by police officers in routine traffic checks. Oral fluid and blood samples were analyzed using gas chromatography-mass spectrometry (GC-MS) for Delta(9)-tetrahydrocannabinol, amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyethylamphetamine, and methylenedioxyamphetamine. The comparison of GC-MS analysis of oral fluid with the Rapid Stat results for cannabis showed a sensitivity of 85%, a specificity of 87%, and a total confirmation rate of 87%. When compared with serum, the sensitivity of the cannabis assay decreased to 71%, the specificity to 60%, and the total confirmation rate to 66%. These findings were possibly caused by an incorrect reading of the THC test results. Comparison of the Rapid Stat amphetamine assay with GC-MS in oral fluid showed a sensitivity of 94%, a specificity of 97%, and a total confirmation rate of 97%. Compared with serum, a sensitivity of 100%, a specificity of 90%, and a total confirmation rate of 92% was found. The amphetamine assay must, therefore, be regarded as satisfactory.

Recently it has been observed increasing number of drugged drivers in Wielkopolska region. In the period from 2006 to 2009 a total number of 2473 blood samples collected from drivers (2141 from alive suspects and 332 from deceased) and examined for psychoactive agents in the Department of Forensic Medicine Poznan University of Medical Sciences. Positive results were obtained in 68.8% and 22.3% of blood samples taken from living men and women respectively. In the group of deceased 7.3% (male) and 8.4% (female) were found positive. The most frequently detected substance were cannabinoids - 57.8%, then amphetamines and its analogues 18.8%, benzodiazepines 5.8%, opiates 3.4% and cocaine benzoiloecgonine 0.9%. In 13.0% concomitant use of amphetamine and cannabinoids were reported.

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2) > 0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

In recent years, there has been observed an increasing number of traffic users being under influence of psychoactive substances that affect the central nervous system. A total of 198 blood samples and 23 urine samples collected from traffic users (drivers, passengers and pedestrians) suspected of having ingested psychoactive substances were examined. The analysis included blood samples collected from living individuals and blood or urine from the deceased. Ethyl alcohol levels were determined by gas chromatography, while body fluids were examined by Elisa tests for determination of cannabinoids, amphetamines, opium narcotics, cocaine (benzoiloecgonine), benzodiazepines, barbiturates and tricyclic antidepressants. The confirmation of positive results was carried out by gas chromatography with mass detector. Twenty-nine blood samples were positive, what constituted 14.6% of the total number of investigated cases, including 12 (7.8%) of samples originating from living individuals and 17 (37.8)--from the fatalities. In both groups, the most commonly detected substances were cannabinoids (THC and its metabolite carboxy-THC) and amphetamines and its analogues.

In the present study, the effects of amphetamine-class drugs were examined in cases reported to the Victorian coroner from 2001 to 2005 to determine if death can occur from the use of amphetamine-class drugs alone. A total of 169 cases were reviewed where a forensic autopsy detected amphetamine(s) in the blood. Pathology, toxicology, and police reports were analyzed in all cases to ascertain the involvement of amphetamine-class drugs in these deaths. In Victoria, methamphetamine (MA) is the principal abused amphetamine-class followed by methylenedioxymethamphetamine (MDMA). There were six cases in which a cerebral hemorrhage caused death and three cases in which serotonin syndrome was established as being caused by the interaction of MDMA and moclobemide. There were 19 cases in which long-term use of amphetamines was associated with heart disease. There were three cases where amphetamine-class drugs alone were regarded as the cause of death, of which two cases exhibited high levels of MDMA and lesser amounts of MA and/or amphetamine. There were no cases in which significant natural disease was absent and death was regarded as caused by the use of MA. There was no correlation between blood concentration of drug and outcome.

We have demonstrated that, with simple pH adjustment, volatile drugs such as methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), ketamine, and valproic acid could be analyzed rapidly from raw biofluid samples (e.g. urine and serum) without dilution, or extraction, using atmospheric pressure ionization. The ion source was a variant type of atmospheric pressure chemical ionization (APCI) that used a dielectric barrier discharge (DBD) to generate the metastable helium gas and reagent ions. The sample solution was loaded in a disposable glass pipette, and the volatile compounds were purged by nitrogen gas to be reacted with the metastable helium gas. The electrodes of the DBD were arranged in such a way that the generated glow discharge was confined within the discharge tube and was not exposed to the analytes. A needle held at 100-500 V was placed between the ion-sampling orifice and the discharge tube to guide the analyte ions into the mass spectrometer. After pH adjustment of the biofluid sample, the amphiphilic drugs were in the form of a water-insoluble oil, which could be concentrated on the liquid surface. By gentle heating of the sample to increase the evaporation rate, rapid and sensitive detection of these drugs in raw urine and serum samples could be achieved in less than 2 min for each sample., (Copyright 2009 John Wiley & Sons, Ltd.)

Our aim was to describe the incidence and trends of driving under the influence of drugs (DUID) and to examine the main drug findings and their trends in suspected DUID cases in Finland. A register-based study was conducted of all suspected DUID cases during 1977-2007. The data included 31,963 DUID offenders apprehended by the police with a positive finding for illicit/licit drug impairing driving performance. Toxicological results were analyzed in blood and/or urine specimens in one central laboratory. The incidence of suspected DUID cases increased 18-fold during 1977-2007. Most of the suspects were men (89.7%). However, the male-female ratio decreased from 13.9 to 7.3. The mean age decreased from 36.2 years in 1977 to 29.9 years in 2001 but has since reincreased. Most often found substances were benzodiazepines (75.7%), amphetamines (46.0%), cannabinoids (27.7%) and opioids (13.8%). Most common illicit drugs, amphetamines and cannabinoids, started to appear at the end of the 1980s. Poly-drug findings were common (77.1%). Suspected DUID cases have increased sharply after the introduction of a zero tolerance law, especially in regard to amphetamines. DUID is an increasing problem in Finland, and needs serious attention.

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.

The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50-5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and -9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.

A two-step auto-injector has been developed for the automated on-column derivatization and subsequent GC-MS of amine-type drugs and metabolites. To effectively derivatize such analytes, this injector has been designed to inject the derivatization reagent several seconds after the sample has been injected. Eleven kinds of amphetamine-type stimulants (ATS) and their typical metabolites were examined, using the trifluoroacetylation reagent N-methyl bis(trifluoroacetamide) (MBTFA). Although the quantitative derivatization of the hydroxyl groups was difficult, this technique was successfully applied to the determination of ATS in urine, blood, and hair specimens. The detection limits of methamphetamine and amphetamine in hair were 0.2 and 0.1 ng/mg hair, respectively, in the full-scan mode, when a 10 mg hair sample is analyzed.

Amphetamines are considered as narcotics in France. Their use induces modifications of the central nervous system and of the cardiovascular, respiratory and urinary systems by a sympathomimetic indirect effect. Here is reported the observation of a young woman who absorbed amphetamines causing a cardiogenic shock on a ground of acute myocarditis. The constitution of haemodynamic, respiratory and neurologic distresses lead to the endotracheal intubation of the patient. The haemodynamic status remaining shaky, despite the use of vasoactive drugs, a circulatory assistance by intra-aortic counter pulsation balloon was carried out. The initial echocardiography showed a left ventricular ejection fraction lower than 20%. Amphetamine's toxicity mechanisms still remain complicated; on cardiovascular plan, some cases of coronary artery spasm have been described. The coronarography, not accomplished immediately, was normal. Toxicological samples revealed an abnormally high amphetamines concentration. The severity of the cardiac attack was amplified by a Mycoplasma pneumoniae myocarditis. There was a positive evolution in eight days. Intoxication and infection can difficultly be dissociated in this case of cardiogenic shock.

Two hundred and forty-seven serum samples which have been collected by police during roadside testing and have been found positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDE) were analyzed for gamma-hydroxybutyrate (GHB). Serum samples were spiked with deuterated GHB as internal standard and acetonitrile was added to achieve dilution and protein precipitation. Samples were analyzed with a LC-MS/MS system operated in the multiple reaction monitoring mode (MRM) using a TurboIonSpray source. Chromatographic separation was achieved using a Synergi Polar RP column applying a gradient elution with a runtime of 15 min. To differentiate between endogenous and exogenously administered GHB a cut-off concentration of 10 microg/mL was applied. Five samples exceeded this concentration and were found positive for GHB. These samples were only found positive for amphetamine but no other amphetamine derivatives were detected, while in three samples THC and in one sample cocaine, benzoylecgonine and ethanol were found.

Innovative features and technical improvements in modern bench-top quadrupole gas chromatograph-mass spectrometer (GC-MS) have prepared the way for faster and more cost-effective applications while still maintaining sufficient chromatographic resolution, speed of MS data acquisition and reliability of analytical methodology. In this paper, a short wide-bore capillary column with low film thickness (5 m x 0.32 mm i.d., 0.1 microm) was used a pre-fractionating column and only chosen heart-cuts were transferred to the second chromatographic dimension (15 m x 0.25 mm i.d., 0.25 microm) by means of a pressure-adjusted continual flow type switching device for quantification of five common amphetamine-type stimulant drugs. The instrumental setting used, in combination with carefully optimized operational fast GC and MS parameters, markedly decreased the retention times of the targeted analytes, e.g., amphetamine 0.891 min and methamphetamine 1.037 min, and the total chromatographic runtime (1.700 min), as well as reducing the need for continuous cleaning of the MS ion source and increasing column life compared with conventional GC-MS approaches. The performance of the instrumental configuration and analytical method was evaluated in validation experiments and the method was also applied to authentic samples. The method demonstrates the potential of fast GC-MS in combination with a gas-phase microfluidic Deans switch device for analysing of (semi)volatile compounds, such as amphetamine-type stimulant (ATS) drugs. This should be particularly useful in modern laboratories aiming at cost-efficient analysis as well as the optimum use of available laboratory capacity and instrumentation.

In this study we reviewed the post-mortem cases in the years 1999-2004 that were presented at the Netherlands Forensic Institute. The concentrations of amphetamine-based drugs in femoral blood from cases of suspected unnatural death were compared with concentrations in whole blood from non-fatal cases of driving under the influence (DUI cases) and with literature. Furthermore, the combinations with other drugs and/or alcohol were investigated. Amphetamine-based drugs were present in 70 post-mortem cases and 467 DUI cases. The most detected amphetamine-based drug was MDMA, followed by amphetamine. The presence of MDA could usually be explained by metabolism of MDMA. Methamphetamine and MDEA were rarely present. Frequently, the amphetamine-based drugs were taken in combination with alcohol and/or other non-amphetamine-based drugs such as cocaine or cannabinoids. The 70 post-mortem cases were divided into 38 amphetamine-based drug caused (i.e. the amphetamine-based drug directly caused or contributed to the death) and 32 amphetamine-based drug related deaths (i.e. death was not directly caused by the amphetamine-based drug). In the latter category, other (poly)drug intoxications and death by violence or drowning were the most frequent causes of death. In 30 cases, MDMA caused death directly. The range in blood concentrations of MDMA in these cases was substantial, i.e. 0.41-84 mg/L with a median concentration of 3.7 mg/L (n=30). MDMA blood concentrations in the MDMA related deaths (n=20) and in the DUI cases (n=360) varied up to 3.7 and 4.0 mg/L, respectively. Seven victims died from the direct effects of amphetamine; the blood concentration of amphetamine ranged from 0.24 to 11.3 mg/L, with a median concentration of 1.7 mg/L (n=7). The median concentrations of amphetamine in the amphetamine related deaths (n=13) and the DUI cases (n=208) were much lower, i.e. 0.28 and 0.22 mg/L, respectively. Amphetamine blood concentrations up to 6.0 and 2.3 mg/L were seen in the drug related deaths and DUI cases, respectively. The most frequently encountered amphetamine-based drugs in the investigated deaths were MDMA and amphetamine. The majority of MDMA- and amphetamine-caused deaths, i.e. 90% of these deaths, occurred with blood concentrations above 1.5 and 0.80 mg/L, respectively. MDMA and amphetamine blood concentrations in drug related deaths and DUI cases, however, overlap the range of fatal concentrations. Therefore, MDMA or amphetamine concentrations should never be used alone to establish the cause of death.

Metamphetamine (MA) is one of the most frequently encountered abused drugs in Japan and the Triage immunoassay kit is often used to screen for this drug. However, immunoassay screening also gives positive results with other structurally related compounds, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA), an ephedrine metabolite and beta-phenethylamine (PEA). Therefore, it is important to develop a simple and reliable method which can determine these drugs simultaneously. This paper describes a simple method for simultaneous identification and quantification of 13 amphetamine related drugs in human whole blood. The method consists of a solid phase extraction using a new polar-enhanced Focus column followed by acetylation and gas chromatography-mass spectrometry in the scan mode. Tetradeuterated MA and trideuterated methylephedrine (ME) were used as internal standards. As the Focus column required only simple extraction steps and provided a clean extract, identification of each drug was feasible even at low concentrations. The calibration curves were linear over the concentration range from 50 to 5000 ng/ml for all drugs with correlation coefficients that exceeded 0.99. The lower limits of detection of the drugs were 5-50 ng/ml. The absolute recoveries for the drugs were 65-95% and 64-89% at concentrations of 100 and 1000 ng/ml, respectively. Accuracy and precision data were satisfactory when using 2 internal standards. The applicability of the assay was proven by the analysis of blood samples in forensic cases. This method should be most useful for confirmation of positive immunoassay results for amphetamines and related drugs.

This study investigated amphetamine concentrations in both oral fluid and whole blood samples of persons suspected of driving under the influence of drugs. The data for the study were obtained from 153 cases. The mean volume of oral fluid collected with the Intercept oral fluid collection device was 224 microL. Because of the small sample volume of oral fluid, the results of the amphetamine concentrations in oral fluid were not used in the calculations for 39 cases. The total number of cases positive for amphetamine in oral fluid was 100 out of 114. In seven cases the oral fluid sample was positive (cutoff 25 microg/L), even though the whole blood sample was negative (cutoff 20 microg/L). All of the cases found positive in whole blood (n = 93) were also positive in oral fluid. Oral fluid would therefore be well suited as a testing matrix for amphetamine when driving under the influence is suspected. The results nevertheless indicated that the cutoff used for amphetamine in oral fluid (i.e., 25 microg/L) could be higher to correspond to the window of detection given by the level of 20 microg/L in whole blood.

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.

An extraction and determination method of most important amphetamine derivatives in serum has been developed. The procedure comprises liquid-liquid extraction with tert-butyl methyl ether of the sample under basic conditions, centrifugation, formation of hydrochloric salts after the separation of organic phase, vacuum evaporation of the organic solvent at 60 degrees C, and trifluoroacetylation by on-line flash injection with MBTFA. GC analysis was performed by electron impact GC-MS in SIM mode. In this way satisfactory identification of 12 amphetamine derivatives could be obtained. Amphetamine, methamphetamine, MDA, MDMA and MDEA could be analyzed by using pentadeuterated analogs as internal standards. Low limits of detection 2.5-6.9 ng/mL could be reached. The assay was linear within the 5-100 ng/mL range with a regression coefficient greater than 0.999 for each compound. Our derivatization method is of low cost since only 1 microL of MBTFA is used for each flash trifluoroacetylation.

A collaborative study was conducted in France in order to determine the prevalence of cannabinoids, opiates, cocaine metabolites and amphetamines in blood samples from drivers killed in road accidents in 2003 and 2004 and to compare these values with those of a previous study performed during the period 2000-2001 involving 900 drivers. Blood samples were provided from 2003 under 30-year-old drivers, killed in a traffic accident. Drugs of abuse were determined by gas chromatography-mass spectrometry using the same analytical procedures in all the 12 laboratories. The most frequently observed compounds were by far cannabinoids, that tested positive in 39.6% of the total number of samples. Delta9 tetrahydrocannabinol (THC), the most active of the principle constituents in marijuana (cannabis sativa), was detected in the blood of 28.9% drivers and was the single drug of abuse in 80.2% of the positive cases. It was associated with amphetamines in 7.4% and with opiates and cocaine in 1.9 and 4.8%, respectively. Amphetamines were present in 3.1% of the total number of samples, cocaine metabolites in 3.0% and opiates in 3.5%. When comparing these results with those of a previous study performed 3 years before, a significant increase is observed for THC (28.9% versus 16.9%), cocaine metabolites (3.0% versus 0.2%) and amphetamines (3.1% versus 1.4%). This study demonstrates the critical necessity of implementing in France as soon as possible systematical roadside testing for drugs of abuse.

Toxicologic analysis is an integral component in the investigation of suicide and requires correlation with a detailed scene inspection, with an extensive exploration into the decedent's medical and social background to uncover suicidal ideation or intent and a postmortem examination of the body. In this review, the authors analyzed 2864 cases classified as suicide upon autopsy and toxicologic examinations between 1993 and 2002 in the Kentucky Division of Medical Examiner's Services. Blood and urine were collected in 95.0% and 72.3% of cases, respectively. A total of 32.5% of the victims had negative blood toxicologic results, and 52.7% of urine toxicology screens yielded no drugs. Analysis of the data indicated that 3 times as many women had taken antidepressants and more than twice as many had consumed opioids. Drug toxicity ("overdose") ranked as the third (9.9%) leading cause of suicide after firearm injury (67.5%) and hanging (13.7%). Women succumbed to drug toxicity more than men (27.5% versus 5.9%). Of the overdose deaths, 66.5% had a negative blood alcohol concentration (BAC), while antidepressants, opioids, and benzodiazepines were detected in blood in 54.4%, 37.4%, and 29.2% of the subjects, respectively. The collection of these data serves the goals of public health and clinicians in devising strategies for suicide prevention.

Due to its high efficiency, selectivity, and sensitivity, CE-ESI/MS has evolved as an efficient technique for the drugs and metabolites analysis in biological matrices. However, a sample preparation is mandatory prior to CE-ESI/MS analysis. To achieve fast and simplified sample preparation of plasma samples, protein precipitation (PP) and liquid-liquid extraction (LLE) were used with two injection techniques: hydrodynamic (HD) and electrokinetic (EK) injection. CE-ESI/MS analyses of pharmaceutical compounds and amphetamine derivatives were developed. Detection limits of 1 ppm were reached with PP and HD injection whereas 1 ppb was detected when samples were prepared with LLE and injected by EK. Same experiments were performed for stereoselective determinations in partial-filling mode and detection limits achieved were equivalent to conventional analysis (0.5 ppb per enantiomer). When complex matrices are analyzed, MS signal suppression or enhancement effects are generally not reproducible and could compromise results with ESI. Therefore, matrix effect was investigated in CE-ESI/MS with a commercially available coaxial sheath-liquid ESI interface used as postcapillary infusion system to determine MS signal alterations. Matrix effects were differentially evidenced according to the selected sample preparation. With PP, signal suppression was observed out of the analyses migration window, while for LLE no relevant matrix effect occurred in all experiments.

Introduction: Amphetamine and its analogues belong to the large group of psychoactive compounds. Due to the growing problem of amphetamine poisoning, author decided to review and improve the laboratory diagnostic procedures with regard to forensic toxicology analysis., Material and Methods: Biological material of 8 persons taken during autopsy for routine toxicological tests was used to examine characteristic of biological matrix. Following excludion the presence of xenobiotics during expertise process, material was divided and kept in sealed containers, then analysed after defined time period: 0, 30 and 90 days. Blood, urine, liver, brain and kidney were considered, 96 samples were examined in total. Biological material was analysed using gas chromatography equipped with flame ionization detector (GC-FID), liquid chromatography with diode array detector (HPLC-DAD), and magnetic resonance spectrometer (H NMR)., Conclusions: 1. Workout analytical GC-FID method enable to verify positive initial result of amphetamine and metamphetamine analysis in urine. 2. Biological matrix is composed mainly of low molecular aliphatic organical compounds which cause significant impact in analytical and opinion problem in amphetamine analysis. Its influence determine in proportion as time of biological material self-decomposition and noted cross-reactivity concern both immunological and chromatographic methods (HPLC-DAD). 3. H NMR method may be used to confirm amphetamine presence in biological material for confirmation of positive results obtained using immunological and enzymatic methods used in forensic toxicology.

In recent years, a number of new designer drugs have entered the illicit drug market. The methylenedioxyderivatives of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and simultaneously quantifying 10 2,5-methylenedioxy-derivatives of amphetamine and phenethylamine in human whole blood, using capillary electrophoresis (CE) with diode array detection (DAD). Using an aqueous pH 2.5 phosphate buffer, CE analysis gave peaks with good symmetry and reproducible migration times. Under these experimental conditions, the 10 amphetamines were resolved in 15 min and without interference from biological matrices (blood). Their identification by migration time was confirmed by their UV spectra recorded with a DAD (190-350 nm). The main advantages of the present method lie in its simplicity, clean and reliable extraction from human whole blood and simultaneous detection and quantification by CE-DAD. The applicability of the method was demonstrated by analysis of in vivo rat blood samples. The method was validated according to international guidelines., ((c) 2005 John Wiley & Sons, Ltd.)

The highly sulfated gamma-CD (HS-gamma-CD) is a chiral selector widely used in CE for the enantioseparation of pharmaceutical compounds. This paper investigated different approaches to reduce the stereoselective analysis time of amphetamine (AT) derivatives according to the chiral selector concentration in the BGE. With high HS-gamma-CD concentration, tested analytes were separated in 3.5 min as anionic complexes with short-end injection technique in reversed polarity mode. However, this procedure presented some limitations in terms of efficiency and resolution, excessive Joule heating and poor compatibility with MS detection. With low HS-gamma-CD concentration, compounds were separated as cations. Conventional approaches to reduce CE analysis time demonstrated critical resolution between some analytes. Therefore, the use of the partial-filling technique compatible with MS detection was carried out. Under optimized conditions, the analysis time for the chiral separation of seven AT like compounds was reduced to 6 min. Moreover, sensitivity of CE-MS was sufficient for the determination of ATs in plasma following a simple liquid-liquid extraction.

A new analytical approach, based on derivatization with 2,2,2-trichloroethyl chloroformate and gas chromatography/mass spectrometry (GC/MS), was investigated for qualitative and quantitative analyses of a large range of amphetamine-related drugs and ephedrines in plasma, urine and hair samples. Sample preparation involved alkaline extraction of analytes from biological samples using Extrelut columns, after addition of the internal standard 3,4-methylenedioxypropylamphetamine (MDPA), and subsequent derivatization to produce 2,2,2-trichloroethylcarbamates. GC/MS analyses, in splitless mode using a slightly polar 30-m capillary column, were performed with quadrupole or ion trap instruments. MS acquisition modes were electron ionization (EI) in full-scan or selected ion monitoring (SIM) modes (quadrupole), and full-scan MS or MS/MS modes with chemical ionization (CI) conditions (ion trap). EI spectra of 2,2,2-trichloroethylcarbamates showed variably abundant molecular ions as well as abundant diagnostic fragment ions, both characterized by ion clusters reflecting the isotope distribution of three chlorine atoms in the derivatized molecules. CI spectra showed abundant protonated molecules. Quantitative studies using EI SIM conditions gave recoveries in the range 74-89%, linear response over ranges of 10-2000 ng/mL (plasma and urine) and 0.20-20 ng/mg (hair), with corresponding limits of detection in the ranges 2-5 ng/mL and 0.1-0.2 ng/mg. Potential applications (following full method validation) include clinical and forensic toxicology, as well as doping control., (Copyright 2005 John Wiley & Sons, Ltd.)

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 microl (oral fluid) or 200 microl (blood, serum, urine) of the sample were mixed with 50 microl of alkaline buffer and 500 microl of extraction-derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.

The subject of the research was the analysis of frequency and type of psychoactive substances used, basing on the determinations the blood and/or urine samples, performed in the toxicological laboratory of the Department of Clinical and Industrial Toxicology Jagiellonian University in Kraków in the period from December 2001 to November 2003. From 17,649 performed determinations--45.5% were positive. 50% of the positive determinations were psychoactive substances. The most often psychoactive substance determined was ethyl alcohol (52.86%), next benzodiazepines (17.41%), amphetamines (10.54%), opiates (8.05%), THC (6.87%), barbiturates (3.74%), and occasionally atropine and cocaine. There was observed a variety of mixed, simultaneously taking psychoactive substances, especially ethyl alcohol, opiates, amphetamine derivatives and cannabinoids. The analysis of the occurrence of psychoactive substances in biological samples from patients treated in different hospital departments, others hospitals and ordered by private persons also was performed. In the last two years 369 private patients ordered psychoactive substances determinations and 78 of them were positive.

Enantiomers of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) exhibit different pharmacological properties. This may be important for the interpretation of analytical results. Plasma samples were analyzed using validated negative ion chemical ionization gas chromatography-mass spectrometry procedures. The results for clinical toxicology cases, divided into screening (SCR) and intoxication (ITX) cases, and those of driving under the influence of drugs (DUID) cases were compared. The concentrations of all enantiomers, except R-(-)-MDA and R-(-)- and S-(+)-MA, in the SCR samples were lower than in ITX and DUID samples. Differences between concentrations in ITX and DUID samples were only significant for both enantiomers of AM (DUID higher). These findings suggested impairment in drugged drivers. Different enantiomer ratios (R vs. S) were found for AM between DUID and SCR samples, for MDMA between ITX and SCR samples, and for MDA between DUID and ITX and DUID and SCR samples. Higher MDMA enantiomer ratios in SCR compared to ITX samples are in accordance with a previously described increase of those ratios over time, possibly allowing differentiation of recent from nonrecent ingestion. Pharmacokinetic analysis of a MDMA poisoning yielded elimination half-lives of 6.0 h for R-(-)-MDMA and 4.1 h for S-(+)-MDMA. The enantiomer ratios rose exponentially over time.

This paper describes highly sensitive HPLC methods for the determination of amphetamine (AP) and methamphetamine (MP) in abusers' plasma and hair samples. AP and MP were derivatized with the fluorescent reagent, DIB-Cl, to yield a highly fluorescent DIB-derivatives of AP and MP, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 and 430 nm, respectively. The separation was achieved on an ODS column with isocratic mobile phases composed of acetoniltrile and citrate buffer (55:45, v/v) for plasma samples and of acetonitrile-methanol-citrate buffer (45:20:37.5, v/v/v) for hair samples. The limits of detection were less than 0.87 ng/mL and 0.12 ng/mg in plasma and hair samples, respectively, for both AP and MP. The methods were then applied to the determination of MP and its metabolite AP in plasma obtained from two cases of illegally ingested MP and in one of the cases' hair received later. Case I was treated with dialysis; samples before and after dialysis were analyzed by the described method. After dialysis for 5 h, the total plasma levels of AP and MP decreased from 720 to 190 ng/mL. For case II, MP and AP levels were monitored for 3 days after digestion. Total plasma levels decreased from 57 ng/mL in the day of digestion to 11 ng/mL after 3 days. In hair samples, AP and MP could also be detected in very low concentrations., (Copyright 2003 John Wiley & Sons, Ltd.)

Drug abuse patterns are different due to cultural, social and geographical differences. Methamphetamine (MA) is the most important drug of abuse in Taiwan followed by opiates. Recently, there has been an increase of ketamine and MDMA abuse in disco dancing clubs. Here, we report the patterns of drug abuse by the participants in a metropolitan city disco-dancing club and the general public in Taiwan. The positive rates of common drugs of abuse detected in samples collected from participants in a dancing club were as follows: MDMA, 75.7%; ketamine, 47.0%; MA, 41.6%; opiates, 0%. Marijuana and cocaine were detected at much lower rates (3.4 and 4.7%, respectively). Ketamine and one of the amphetamines were detected together in 42.9% of the samples. The positive rates in samples collected from police detainees suspected of drug abuse in the general public were as follows: MA, 76.0%; OPA, 37.0%; MDMA, 6.0%; ketamine, 2.0%.

The classical stimulants amphetamine, methamphetamine, ethylamphetamine and the amphetamine-derived designer drugs MDA, MDMA ('ecstasy'), MDEA, BDB and MBDB have been widely abused for a relatively long time. In recent years, a number of newer designer drugs have entered the illicit drug market. 4-Methylthioamphetamine (MTA), p-methoxyamphetamine (PMA) and p-methoxymethamphetamine (PMMA) are also derived from amphetamine. Other designer drugs are derived from piperazine, such as benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP), trifluoromethylphenylpiperazine (TFMPP), m-chlorophenylpiperazine (mCPP) and p-methoxyphenylpiperazine (MeOPP). A number of severe or even fatal intoxications involving these newer substances, especially PMA, have been reported. This paper describes a method for screening for and simultaneous quantification of the above-mentioned compounds and the metabolites p-hydroxyamphetamine and p-hydroxymethamphetamine (pholedrine) in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. It was linear from 5 to 1000 micro g l(-1) for all analytes. Data for accuracy and precision were within required limits with the exception of those for MDBP. The limit of quantification was 5 micro g l(-1) for all analytes. The applicability of the assay was proven by analysis of authentic plasma samples and of a certified reference sample. This procedure should also be suitable for confirmation of immunoassay results positive for amphetamines and/or designer drugs of the ecstasy type., (Copyright 2003 John Wiley & Sons, Ltd.)

Four commonly used immunoassay kits were evaluated for their efficiency in screening for drugs of abuse in whole blood. Six groups of illicit drugs (opiates, cannabinoids, amphetamines, cocain and benzoylecgonine, benzodiazepines, and methadone) were determined by using the homogenous assays ADx and CEDIA DAU and compared with the results produced by means of the inhomogenous assays MTP and Pyxis 24. The measured 86 blood samples were taken from authentic routine analyses between February and September, 2000. Chromatographic confirmation analyses were carried out in all cases (positive and negative immunochemical pretesting). The cutoff levels were retrospectively optimized to reduce false-negative results with priority. Furthermore, false-positive pretests were minimized in order to decrease laboratory work under economical aspects. Specificity and sensitivity were determined for each parameter and assay. For the ADx assay, specificities of 54% (cannabinoids) to 97% (cocaine and metabolite) and sensitivities of about 67% (amphetamine class) to 94% (opiates) were found. The CEDIA assay revealed specificities of 77% (methadone) up to 100% (benzodiazepines) and 75-96% sensitivities for amphetamines and opiates. The MTP immunoassay resulted in specificities of 52% (methadone) to 95% (opiates, cocain, and metabolite) and sensitivities of 92% (amphetamines) up to 100% (methadone). The evaluation of the Pyxis 24 resulted in specificities of 70-96% (benzodiazepines and amphetamines) and sensitivities of 75% (amphetamines) up to 100% (cannabinoids and methadone), respectively. In conclusion, the microtiterplate immunoassays revealed higher sensitivities and have proved to be at an advantage detecting the lowest concentrations of drugs. However, especially for clinical applications in emergency cases with acute intoxications, when screening results are urgently required, homogenic assays such as ADx or Cedia provide preferable alternatives with faster and easier handling.

In Germany, the substitution of methadone for heroin abusers has arisen recently and has resulted in various medicolegal problems. Normally, these concern methadone-associated deaths, or the prescription of methadone resulting in criminal prosecutions concerning physicians. Not to be forgotten is the problem of driving while taking methadone. In the years 1997-2001, we detected methadone in the blood of 398 cases that were analysed by the Institute of Legal Medicine, Bonn. Methadone was the only drug in only 18 cases. In most of the cases, up to five additional drugs were also being taken: benzodiazepines (61%), ethanol (40%), morphine (39%), cannabinoids (35%), cocaine (28%), anti-depressants (3%), and amphetamines (2%).

A collaborative case-control study was conducted in France in order to determine the prevalence of alcohol, cannabinoids, opiates, cocaine metabolites, amphetamines and therapeutic psychoactive drugs in blood samples from drivers injured in road accidents and to compare these values with those of a control population. Recruitment was performed in emergency departments of six university or general hospitals and comprised 900 drivers involved in a non-fatal accident and 900 patients (controls) who attended the same emergency units for a non-traumatic reason. Drivers and controls were matched by sex and age. Alcohol was determined by flame ionization-gas chromatography, drugs of abuse (DOA) by gas chromatography-mass spectrometry with the same analytical procedures in the six laboratories, and medicines by high performance liquid chromatography with diode array detection. Blood alcohol concentration exceeding 0.5 g/l (i.e. the legal French threshold) was found in 26% of drivers and 9% of controls. In the 18-27 years age range, alcohol was the only toxic found in blood samples of 17% drivers and 5% controls, leading to an odds-ratio (OR) of 3.8. A significant relationship was found between alcohol blood concentrations and OR values. All age groups confounded, the main active substance of cannabis, Delta(9) tetrahydrocannabinol (THC), was found in 10% of drivers and 5% of controls. In the less than 27 years old, THC (>1 ng/ml) was detected alone in the blood of 15.3% drivers and of 6.7% controls, giving OR=2.5, whereas there was no link between THC blood concentrations and OR value. THC was found alone in 60% of cases and associated with alcohol in 32%, with OR=4.6 between drivers and controls for this association. The difference in morphine prevalence between drivers (2.7%) and controls (0.03%) was highly significant (P<0.001), with OR=8.2. The number of positive cases for amphetamines and cocaine metabolites was too low for reaching any interpretation. The most frequently observed psychoactive therapeutic drugs were by far benzodiazepines, that were found alone in 9.4% of drivers and 5.8% of controls, which led to OR=1.7 (P<0.01). This study demonstrates a higher prevalence of opiates, alcohol, cannabinoids and the combination of these last two compounds in blood samples from drivers involved in road accidents than in those from controls, which suggests a causal role for these compounds in road crashes.

Hair and plasma from patients on long-term selegiline medication were analyzed to evaluate the relationships between plasma and hair melanin concentrations and the incorporation of the selegiline metabolites methamphetamine and amphetamine in hair, and to evaluate hair analyses for determining compliance in medication. Analyses were performed on both the whole hairs, as well as pigmented and non-pigmented hairs from gray-haired patients. Melanin was quantitated by spectrophotometry, and metabolites were quantitated by gas chromatography-mass spectrometry. Concentrations in pigmented and non-pigmented hairs differed significantly for both methamphetamine (p < 0.01) and amphetamine (p < 0.02), with mean concentration ratios being 3.69 +/- 1.88 and 2.95 +/- 1.16 for methamphetamine and amphetamine, respectively. Segmental analysis indicated that some patients had not been compliant with medication. We concluded that the incorporation of methamphetamine and amphetamine into hair of single individuals shows a preference for pigmented hairs over white hairs and that segmental analysis of hair may be useful when measuring compliance with medication.

Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatography-mass spectrometry. However, this procedure is labor-intensive and time-consuming, particularly as a preliminary extraction and derivatization are usually unavoidable. Here we describe the development of an alternative method. Amphetamines were isolated from human plasma and oral fluid using a simple methanol precipitation step and subsequently analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. Quantitation of the drugs was performed using multiple reaction monitoring. The developed method, which requires only 50 microL of biological sample, has a total analysis time of less than 20 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 microg/L or better were obtained. The method has been validated and subsequently applied to the analysis of plasma and oral fluid samples collected from current drug users.